Steroids, vitamins, retinoids, and thyroid hormones exert profound effects on cell growth, development, differentiation, homeostasis and tumorigenesis through their cognate nuclear receptors which make up a superfamily of structurally related intracellular hormone-activated transcription factors. Recent evidence suggests that transcriptional regulation by nuclear hormone receptors (NHRs) require a diverse group of proteins termed co-activators. Co-activators portray a growing class of proteins that interact with receptors in a hormone-dependent manner and are required for maximal gene activation by the receptors. It is the general belief that co-activators enhance receptor function by acting as a bridge between the DNA-bound receptor and basal transcription factors of the pre-initiation complex and also by providing histone acetyl transferase activity, HAT, which disrupts the local repressive chromatin structure and contributes to increase transcriptional activity. Recently, our laboratory and other have cloned ubiquitin-proteasome and ubiquitin-like (NEDD8, neural precursor cell-expressed developmentally down-regulated) pathway enzymes, E6-associated protein (E6-AP), RSP5/RPF1, ubiquitin- conjugating enzymes (UBCs), SUG1 and E1 ubiquitin-activating enzymes (UBA3) as co-activators of NHRs. These co-activators possess other enzymatic activities such as ubiquitin activation, ubiquitination conjugation, ubiquitin ligation and protease activities, instead of HAT activity. These observations suggest a possible regulatory role for the ubiquitin and NEDD8 pathways in nuclear hormone receptor-mediated gene activation. However, the exact mechanisms by which these pathways regulate nuclear receptor gene activation is completely unknown at this time. Based on our preliminary observations, we hypothesize that the ubiquitin and NEDD8 pathways are modulators of nuclear hormone receptor co-activation. In order to understand the mechanism by which these pathways modulate nuclear hormone receptor gene activation, we propose the following three specific aims: A) To identify and characterize the E6-AP interacting protein(s) and study their role along with E6-AP in nuclear hormone receptor co-activation, B) To identify and characterize target proteins of the ubiquitin-proteasome pathway and understand the mechanism by which these pathways modulate nuclear hormone receptor gene activation, we propose the following three specific aims: A) To identify and characterize the E6-AP interacting protein(s) and study their role along with E6-AP in nuclear hormone receptor co-activation, B) To identify and characterize target proteins of the ubiquitin-proteasome pathway and understand the mechanism by which this pathway modulates nuclear hormone receptor gene activation, and C) To characterize the E1 ubiquitin-activating enzyme, UBA3,. Of the NEDD8 pathway as a co-activator of NHRs and to characterize this role in nuclear hormone receptor co-activation.